The present invention relates generally to obtaining a natural mixture of conjugated estrogens from the urine of pregnant mares, and specifically to the separation of cresol from mares' urine.
Estrogens are used in medicine for hormone replacement therapy. In particular, estrogen mixtures are used for the treatment and prophylaxis of the disorders of the climacteric period which occur in women after natural or artificial menopause. In this case, natural mixtures of conjugated estrogens such as are found in the urine of pregnant mares have proved particularly effective and readily compatible.
The dissolved solids content in the urine of pregnant mares (=pregnant mares' urine, abbreviated hereafter as “PMU”) may naturally vary within wide ranges, and may generally lie in a range of 40–90 g dry substance per liter. In addition to urea and other usual urine contents, phenolic constituents are contained in the solids content of the PMU in quantities of about 2–5% by weight relative to dry matter. These phenolic constituents include cresols and dihydro-3,4-bis[(3-hydroxyphenyl)methyl]-2(3H)-furanone, known as HPMF. These may be present in free or conjugated form. The PMU contains a natural mixture of estrogens which is largely present in conjugated form, e.g. as sulfuric acid semi-ester sodium salt (abbreviated hereafter as “sulfate salt). The content of conjugated estrogens (=conjugated estrogens, abbreviated hereafter as “CE”), calculated as estrogen sulfate salt and relative to dry matter, may be between 0.3 and 1% by weight.
Various procedures for the direct processing and obtention of the conjugated estrogens contained in the PMU are described in the prior art. Usually extracts containing conjugated estrogens are obtained from the PMU by extraction with a polar organic solvent which is not miscible, or only slightly miscible, with water, such as ethyl acetate, n-butanol or cyclohexanol. With such liquid-liquid extractions, however, a number of problems occur, such as severe foaming, sedimentation, emulsification and poor phase separation. Generally several extraction steps are required, which results in losses and only partial obtention of the estrogen content. To avoid these disadvantages, therefore, a number of solid-phase extraction methods have been proposed in the prior art.
Heikkinnen et al. (Clin. Chem. 27/7, (1981), 1186–1189) and Shackleton et al. (Clinica Chimica Acta 107 (1980), 231–243) describe a solid-phase extraction of estrogens by means of a cartridge with silanized silica gel containing octadecylsilane radicals (Sep-PakR C18 cartridge, manufactured by Waters Ass. Inc. Milford, Mass., USA) for the processing of small quantities of urine and plasma for analytical determination of estrogens by means of gas chromatography. Therein, the estrogens are eluted from the cartridge with methanol.
In 1968, H. L. Bradlow proposed [see Steroids 11:265–272 (1968)] to use Amberlite XAD-2R, a neutral, non-polar hydrophobic polystyrene resin, manufactured by Rohm und Haas, for the extraction of conjugated estrogens from urine. The adsorption capacity quoted is low. According to Bradlow, an optionally diluted urine is passed through a column containing the resin at a low throughflow rate. The estrogens are eluted with methanol or ethanol.
More recent patent applications describe methods for obtaining an extract containing the natural mixture of conjugated estrogens from mares' urine by solid-phase extraction of the mixture of conjugated estrogens from the urine of pregnant mares e.g. on RP silica gel (WO 98/08525) or on non-ionic semipolar polymeric adsorption resins (WO 98/08526). The methods described in these international patent applications still use PMU starting materials with relatively high proportions of phenolic urine contents such as cresol and HPMF, which, although they have already been able to be separated out successfully by the method described, on the other hand limit the efficiency of the actual working-up as undesirable accompanying substances, e.g. by reducing the capacity of the respective adsorbent.
Depending on the origin of the urine or feed of the horses, a cresol content of more than 500 mg/l up to occasionally even above 1,500 mg/l may also be recorded in fresh native urine. The cresol content additionally increases according to the origin, age of the urine, degree of bacterial contamination and the storage conditions, in particular e.g. according to the storage temperature, quite conceivably to values of up to 2,000 mg/l and possibly even more. Although reliable separation of cresol is ensured for example by the alkaline washing of the adsorber column with non-ionic semipolar polymeric adsorption resins in the method in accordance with international patent application WO 98/08526, a high cresol content is disruptive for two reasons:    a) since cresol is adsorbed by the resin in addition to the hormones, the hormone capacity of the resin decreases; and    b) at high cresol contents in the alkaline washing stage, a hormone loss of several percent is to be expected during this step.
In order to avoid these disadvantages, it is desirable to process urine with as low a cresol content as possible. Since low cresol contents in the PMU cannot be ensured for reasons of transportation—e.g. when the PMU is collected throughout the world, even in out-of-the-way regions—or also because of the breed of and feed given to the horses, other measures must be sought to minimize the cresol content in the PMU before the working-up and recovery of the mixtures of conjugated estrogens. Tests hitherto, e.g. adsorption tests, were not successful, since a reduction in cresol was accompanied by more or less great, unacceptable hormone losses.
In addition to the optimization, described in the prior art, of the direct, complete working-up of pregnant mares' urine (PMU) to obtain natural mixtures of conjugated estrogens (CE), therefore, also the steps preceding working-up, such as ensuring estrogen-protecting removal of undesirable accompanying substances such as cresols, are also of particular significance for effective working-up and obtaining of a high-quality and -quantity mixture of conjugated estrogens.